Mutagens from the cooking of food. III. Survey by Ames/Salmonella test of mutagen formation in secondary sources of cooked dietary protein

The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA—ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon—were examined for their mutagenicity towards S. typhimurium TA1538 after normal ‘household’ cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling at 100–475°C). Well-done fried ground beef, beef steak, ham, pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs and egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperatures above 225°C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Nephrotoxicity of cis-diamminedichloroplatinum (II) (cis-platinum) and the additive effect of antibiotics: Morphological and functional observation in rats

Basic morphological and functional patterns of cis-platinum nephrotoxicity and the additive effect of combined antibiotics were investigated in male Wistar rats. Nephrotoxicity by cis-platinum is produced in a dose-related fashion and characterized by tubular damage primarily involving the corticomedullary junction. Partial recovery of renal function accompanied by diuresis occurs in 2–3 weeks, however, glomerular filtration rate (GFR) remains at low levels. Histologically, patchy areas of cystic changes remains in the inner cortex, among the areas of relatively normal nephrons. Concomitant use of antibiotics such as aminobenzyl penicillin, sulbenicillin, and cephalothin had no additive effect on platinum nephrotoxicity, from morphological and functional aspects. However, a combination of tobramycin and cis-platinum induced severe renal damage with most animals showing a continuous elevation of blood urea nitrogen and decrease in body weight before death. Histologically, acute tubular necrosis involving both inner and outer cortices was evident. Although cis-platinum is clinically used under enhanced diuresis, GFR and tubular damage have to be carefully monitored throughout the course of cis-platinum treatment. When nephrotoxic antibiotics are being concomitantly prescribed for patients already ingesting cis-platinum, then special precautions should be taken.     

Dose-response studies of gentamicin nephrotoxicity in rats with experimental renal dysfunction. II. Polyvinyl alcohol glomerulopathy

Animal studies involving concurrent pathophysiologic states, including experimental renal dysfunction, are useful for a proper understanding of the mechanisms of gentamicin nephrotoxicity and acute renal failure. This study examined gentamicin nephrotoxicity in a model of glomerular dysfunction in rats. Administration of medium molecular weight polyvinyl alcohol (PVA) produced a glomerulopathy, with characteristic accumulation of macromolecular PVA in the glomerular mesangium without altering glomerular filtration or causing proteinuria. Subsequent daily doses of gentamicin ranging from 0 to 120 mg/kg elicited a dose-response nephrotoxicity in both control and PVA-pretreated rats after 6 or 12 days of drug. Based on statistical analysis of renal clearances of creatinine, urea, sodium, and potassium; serum creatinine and urea nitrogen; urinary N-acetyl-beta-D-glucosaminidase excretion (6 days only); in vitro renal cortical transport of tetraethylammonium (TEA) (6 days); and quantified light-microscopic data (12 days), PVA induced an early (6 days) sensitivity to gentamicin nephrotoxicity. By 12 days, there were no differences in the responses of control and PVA rats to gentamicin. Single-dose gentamicin clearance, volume of distribution, and half-life were not altered by PVA and renal concentrations at 6 days were generally lower in these rats. Results of TEA transport studies tend to rule out PVA-induced metabolic lesions in the proximal tubular epithelium as the mechanism for the early sensitivity. This investigation demonstrates altered gentamicin nephrotoxicity in rats with an otherwise benign glomerulopathy and, combined with similar conclusions from a related study in subtotally nephrectomized rats, presents further evidence that the underlying pathophysiologic state of the kidney is an important factor in the renal response to nephrotoxins.     

Effects of pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin on the capacity of hepatic and extrahepatic mouse tissues to convert procarcinogens to mutagens for Salmonella typhimurium auxotrophs.

Using the conventional Ames test, the conversion of three procarcinogens, 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BaP), and N-2-fluorenylacetamide (FAA) to intermediary metabolites mutagenic to Salmonella typhimurium tester strains TA-98, TA-100, but not TA-1538 was much more efficient in hepatic S-9 fractions prepared from TCDD-pretreated mice than in analogous fractions from untreated mice. In general, renal and pulmonary S-9 fractions from TCDD-pretreated mice also generated more back mutations than the corresponding controls however, statistical significance could not be demonstrated in every case. Exceptions were with DMBA, for which pulmonary S-9 fractions of TCDD-pretreated mice produced fewer mutations than corresponding fractions from control animals and with FAA, for which renal S-9 fractions of pretreated mice produced approximately the same numbers of mutations as fractions from control animals. Quantitation of the generation of metabolites cytotoxic to bacteria grown on nutrient agar revealed that TCDD pretreatment resulted in increases in the capacity of S-9 fractions from mouse hepatic tissue to generate cytotoxic metabolites from BaP and FAA but not from DMBA. The kidney was more responsive than the liver or lung to monooxygenase induction by the dioxin with BaP or DMBA as substrates but analyses of metabolite profiles with hplc indicated that TCDD pretreatment appeared to produce no qualitative differences in metabolites generated in the tissues studied. Differences of ratios of metabolites were noted with respect to DMBA metabolites cochromatographing with 7-hydroxymethyl-12-methylbenz(a) anthracene and trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz(a)anthracene. Pretreatment of mice with TCDD produced 2- to 10-fold increases in the amounts of these two metabolites in comparison with slight to moderate increases in quantities of most other metabolites and a decrease in those eluting with 12-hydroxymethyl-7-methylbenz(a)anthracene. The effect was particularly prominent in the kidney. In general, the data indicated an overall positive correlation between the activity of tissue monooxygenases and capacity of the same tissues to metabolically convert procarcinogens to mutagens in the Ames system.     

Sulfate absorption from the airways of the isolated perfused rat lung.

The kinetics of sulfate absorption from the airways of the isolated perfused rat lung are presented. Absorption of sulfate ion appears to be by simple diffusion and to be enhanced in the presence of ammonium ion at low concentrations. The $\text{t}\text{1}\text{2}$ for the initial rate was 11.9 ± 1.4 min. The administration of 1 μmol of (NH₄)₂SO₄ intratracheally led to a rapid decrease in the respiratory volume of the lung, an effect which could be blocked by prior perfusion with mepyramine maleate (10^?5m). Ammonium sulfate caused a rapid release of a large portion of the histamine stores into the lung perfusate. The results suggest that sulfate compounds are absorbed rapidly by the lung and that histamine release may occur during absorption. The biological effects of sulfate-containing aerosols may be related to the liberation of histamine reserves.     

Capillary permeability-increasing enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi): activity due to the release of peptide material from a protein in bovine plasma

When a mixture of the purified capillary permeability-increasing enzyme from A. caliginosus venom and bovine plasma or heated bovine plasma was injected into the depilated skin of the back of a rabbit, the capillary permeability-increasing activity was much greater than that induced by injection of the enzyme alone. The substance which increases capillary permeability was extracted from the incubated mixture of bovine plasma and enzyme with 50 – 70% ethanol. Its activity was lost when treated with carboxypeptidase A. Thus, it is supposed that the increase in capillary permeability induced by the enzyme is due to a low molecular weight peptide released from a protein in bovine plasma by the action of the enzyme. No liberation by the enzyme of histamine or anaphylatoxins of the complement system was found.     

The effects of a toxin (ATX II) from the sea anemone Anemonia sulcata on the electrical and mechanical activity of the denervated hemidiaphragm of the rat

Isolated strips of rat diaphragm denervated 9 – 21 days prior to experimentation were used to study the effects of the sea anemone toxin ATX II on electrical and mechanical activity in mammalian skeletal muscle. ATX II increased twitch tension transiently and induced a concentration-dependent rise in muscle tone. The resting membrane potential was increased to more negative values by low concentrations of ATX II (? 10^?7M) and was decreased only with high concentrations (? 2 × 10^?7M). ATX II at 10^?7M) prolonged the action potential duration, however, individual fibres exhibited a large variation in response. Lidocaine (5 × 10^?5M) reversed the toxin-induced contracture, but did not inhibit the ATX II-induced prolongation of action potential duration. ATX II increased the incidence of spontaneous action potentials which occur in denervated skeletal muscle. This effect was partially reversed by tetrodotoxin (10^?5M), which also partially abolished the ATX II-induced contracture. Reduction in toxin-induced spontaneous activity was also observed in the presence of lidocaine (5 × 10^?5M) or acetylcholine (10^?5M). Both agents diminished ATX II-induced contracture. It is concluded that the increased muscle tone observed with ATX II may reflect a summation of increased fibrillatory activity.     

Studies of the molecular pathogenesis of hexane neuropathy: II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking

In the reaction between ethanolamine and 2,5-hexanedione, 1-(2-hydroxyethyl)-2,5-dimethylpyrrole was formed, and the pyrrole was found to autoxidize to form an orange chromophore. Similar orange chromophores were observed in the reaction of 2,5-hexanedione, 2,5-heptanedione, and 3,6-octanedione with a variety of primary amines and with proteins. The development of the orange chromophore in the reaction of 2,5-hexanedione with proteins was attended by a proportional derivatization of lysyl residues and by extensive intramolecular and intermolecular crosslinking. These observations suggest that the sequence of events in the crosslinking of neurofilaments during chronic n-hexane intoxication may be metabolism to 2,5-hexanedione, formation of an imine with lysyl residues, cyclization to form a pyrrole, autoxidation of the pyrrole, and finally covalent crosslinking involving pyrrole rings.     

Transplacental induction of mixed-function oxygenases in extra-hepatic tissues by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.     

Modulation of pulmonary p-nitroanisole O-demethylase by intratracheally instilled heavy metal salts

Isolated, ventilated, and perfused rat lung preparations were used to investigate the acute effects of intratracheally applied heavy metal salts on the O-demethylation of p-nitroanisole (NA). The NA O-demethylase activity of the lung was characterized by induction (104%) following pretreatment of rats with β-naphthoflavone (BNF, 80 mg/kg), and by inhibition with SKF-525A (50 μm), metyrapone (0.1 mm), or ventilation of the lungs with CO. Pretreatment of rats with indomethacin (25 mg/kg × 3 days, po) or 3-amino-1,2,4-triazole (1 g/kg, ip) did not alter this activity. The NA O-demethylase was, therefore, suggested to be mixed function oxidase (MFO). Intratracheal instillation of NiCl₂ (0.1 or 1.0 μmol/lung) inhibited the O-demethylase activity (30 or 54%, respectively). The apparent K_m of the reaction (0.138 mm) was doubled by NiCl₂ treatment (0.252 mm), and the V_max was decreased from 26.8 to 20.9 nmol p-nitrophenol/min/g. Cadmium chloride (1.0 μmol/lung) increased the activity by 80%, but the K_m was unchanged. No effect was observed at a dose of 0.1 μmol CdCl₂/lung. At doses of 0.1 or 1.0 μmol/lung, CoCl₂ was without measurable effect on this activity. These data suggest that heavy metals present in the atmosphere can interact with pulmonary MFO in low concentrations to alter the metabolism of xenobiotic compounds.     

d-Penicillamine toxicity in mice. III. Pathological study of offspring of penicillamine-fed pregnant and lactating mice

Mice were divided into six experimental diet groups during pregnancy and lactation: (1) C + C, control diet; (2) P(2) + (2), 2 g of d-penicillamine/kg of diet; (3) P(4) + P(4), 4 g of d-penicillamine/kg of diet (these groups received diets throughout pregnancy and lactation); (4) P(4) + P(4)Cu, the test diet was fed during pregnancy, and 5 × 10^?3, m copper sulfate solution was added to replace drinking water after delivery; (5) C + P(4), the test diet was given only during lactation; (6) P(4) + C, the test diet was given only during pregnancy. Highest mortality, most severe and variegated neurological abnormalities, and rupture of aortic aneurysms were observed in the P(4) + P(4) offspring. Supplementation with copper (P(4) + P(4)Cu) resulted in no abnormalities. In C + P(4), mortality was higher and abnormalities were more variegated than in P(4) + C or P(2) + P(2). No abnormality was detected in C + C. The aortas in P(4) + P(4) showed dissecting aneurysms and breaks in the elastic lamellae; neuronal degeneration was noted in the cerebral cortex, thalamic nuclei, and spinal ganglion on the 14th and 21st postnatal days, but not on the 1st or 7th postnatal days. No neuronal degeneration was observed in the copper-supplemented group. In C + P(4), there were a few abnormal neurons in the cerebral cortex on the 21st postnatal day. No neuronal degeneration was detected in C + C, P(2) + P(2), or P(4) + C. These observations suggest that abnormal signs and gross lesions are related to copper deficiency induced by the metal-chelating action of d-penicillamine especially postnatally through two basic mechanisms: maturation defect in fibrous protein and neuronal degeneration in the brain and spinal ganglia.     

Inhibition of DNA synthesis and alteration to DNA structure by the phenacetin analog p-aminophenol

p-Aminophenol a structural analog and minor metabolite of phenacetin has previously been shown to be a potent nephrotoxic agent. In this report we have shown that p-aminophenol has a marked effect on DNA function and structure. DNA synthesis was inhibited in a dose-dependent manner in human lymphoblastoid cells after exposure to p-aminophenol. Results suggest that DNA synthesis is inhibited by the action of p-aminophenol on DNA structure. At low concentrations of p-aminophenol a reduction in the degree of supercoiling of cellular DNA is observed, as determined by sedimentation under neutral conditions. However at higher concentrations an increase in sedimentation of nucleoids (supercoiled molecules) is obtained which is indicative of an increased level of supercoiling or a more compact structural form of DNA due to folding or aggregation.The number of single strand breaks in DNA, when determined by sedimentation in alkaline sucrose gradients, increases with increasing dose of p-aminophenol. The increase in strand breakage observed at lower concentrations of p-aminophenol agrees with the reduced sedimentation rate obtained under neutral conditions. At higher concentrations of p-aminophenol the extent of breakage of DNA increases under alkaline conditions but an increase in sedimentation occurs under neutral conditions.     

The action of equinatoxin,a peptide from the venom of the sea anemone, Actinia equina, on the isolated lung

On perfusion through isolated lungs from male Sprague-Dawley rats, equinatoxin caused a dose-dependent increase in the wet to dry weight ratio. Ratios were significantly elevated above control values at equinatoxin concentrations of 80–200 ng/ml. The increased ratios were accompanied by an increase in the permeability of the lung vasculature. When equinatoxin was perfused through isolated lungs at concentrations of 100 ng/ml or greater, significantly more [³H]polyethylene glycol (PEG; approximately 900 mol. wt) was retained in the extravascular space as compared to controls. Perfusion pressures of the lung were significantly elevated above controls at equinatoxin concentrations greater than 100 ng/ml. These effects of equinatoxin were not mediated by degranulation of mast cells, as preperfusion of the lung with 100 or 200 μM Na cromolyn or 1 μM lanthanum chloride did not modify the pulmonary response to equinatoxin. At concentrations of equinatoxin below 150 ng/ml the fluid movement appears to be restricted primarily to intracellular, or possibly interstitial, spaces, as no significant amounts of [³H]polyethylene glycol were recovered by tracheal lavage. At concentrations of equinatoxin equal to or greater than 150 ng/ml, significant amounts of PEG were washed from the trachea. As it is a potent inducer of pulmonary edema, equinatoxin may become an important probe to study fluid regulation in the lung.     

A comparison of xenobiotic metabolism in cells isolated from rat liver and small intestinal mucosa.

The metabolism of several foreign compounds has been investigated in viable isolated liver and intestinal mucosal cells. Glucuronic acid and sulphate conjugation was observed in both cell preparations with respect to phenol. Conjugation of 1-naphthol was also observed in isolated intestinal cells. Acetanilide was the only metabolic product of aniline observed with intestinal cells. N-Acetylation was also found to be the major pathway of aniline metabolism in liver cells but p-hydroxylation followed by O-conjugation were also important reactions. Intestinal cells thus appear to be generally effective in conjugation reactions. However, in contrast to hepatocytes the intestinal cells were unable to form glycine conjugates from benzoic acid. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was investigated in isolated intestinal cells from control, 3-methylcholanthrene and phenobarbitone pretreated rats. Glucuronic acid and sulphate conjugation rates of 7-hydroxycoumarin appeared to be unaffected by these pretreatments whereas increases in 7-ethoxycoumarin O-deethylation were observed.     

Neuromuscular and lethal effects of phospholipase A from Vipera ammodytes venom

—Four homogeneous proteins having phospholipase A activity were separated and studied for their i.v. lethal effects in mice and nerve-muscle activity in the guinea pig diaphragm preparation. Fraction “j” had an ld₅₀ of 0.30 mg/kg, with 3.6 μg/ml of bath solution causing a decrease in the indirectly-elicited nerve-muscle contractions to 20% of control, without significantly changing the directly-elicited muscle contractions. Fraction “k₂” had an ld₅₀ of 0.021 mg/kg and caused a similar nerve or nerve-muscle block at 5 μg/ml of bath solution, without altering the directly-elicited contractions. Fraction “k₁” had an ld₅₀ of 0.58 mg/kg and produced less distinct but dose-related changes in the nerve or in nerve-muscle transmission, as well as weakening directly-elicited muscle contractions to within 60% of control. Fraction “l” had an ld₅₀ of 3.6 mg/kg but high doses of the fraction were required to produce changes in diaphragm contractility. High doses (60 and 195 μg/ml) produced marked effects on both the directly- and indirectly-elicited contractions, suggesting that this fraction affects the muscle directly.     

Hydrolysis of ester- and amide-type drugs by the purified isoenzymes of nonspecific carboxylesterase from rat liver

Five purified carboxylesterases from rat liver microsomes show a differing capacity for the hydrolysis of ester- and amide-type drugs. The two closely related enzymes that are responsible for the microsomal hydrolysis of palmitoyl-CoA and long chain monoacylglycerides exhibit the highest propanidid-and aspirin-cleaving rates. The predominant nonspecific esterase of microsomes is responsible for the hydrolysis of procaine, clofibrate, isoarecaidine esters, butanilicaine, octanoylamide, and possibly butyryl thiocholine. Finally, the palmitoyl carnitine-cleaving esterase splits phenacetin and acetanilide. The purified nonspecific esterase with the lowest isoelectric point is not involved in the metabolism of the drugs mentioned.     

Ichthyotoxism by a paralytic toxin produced by marine dinoflagellates of the genus Ceratium: relationship to fraction beta isolated from the sponge Tedania ignis

Two hundred tons of the plankton feeding sardine Cetengraulis edentulus died in March 1982, in Carenero (10 degrees 10' N, 66 degrees 05' W), Venezuela. A fraction was extracted from this fish that was toxic to mice by i.p. injection. The animals died in less than 4 min and showed generalized flaccid paralysis. Gel filtration on Sephadex G15 and Bio Gel P2 showed that the toxicity is related to a fraction that blocks the release of acetylcholine in frog (Rana pipiens) neuromuscular junctions. This toxin is similar in chemical properties and presynaptic effect to fraction beta isolated from the sponge T. ignis by Sevcik and Barboza. Fractions of the same biological action and chemical properties were isolated from plankton samples collected in the area of the ichthyotoxism. The correlation analysis between the presence of toxin and a plankton species in a sample, was carried out with a feasibility index (as %) defined by Sevcik and Mijares. Random samples of plankton (29) were collected in 3 locations (11 degrees 50' N, 68 degrees 15' W; 10 degrees 36' 24' N, 67 degrees 14'7' W and 10 degrees 21' N, 64 degrees 21' W). The correlation carried out over 167 species of phytoplankton present in the samples indicates that the species most likely to be responsible for the production of the fraction beta, in order of feasibility index (in parentheses) are: Ceratium furca (54%), Protoperidinium sp. (1.7%) and Protoperidinium pallidum (1.6%). In some samples a fraction similar to fraction alpha from T. ignis was also found. The identification of the phytoplankton responsible for this fraction is, however, less conclusive. The feasibility indexes are: Protoperidinium sp. (58%), Ceratium inflatum (30%), Podolampas sp. (23%), Ornithocercus steini (21%). The genus Ceratium was the second most abundant in Carenero at the time of the fish death. These results suggest that the toxins isolated from C. edentulus and T. ignis have a planktonic origin.     

Glutathione adduct formation with microsomally activated metabolites of the pulmonary alkylating and cytotoxic agent, 3-methylindole

Incubations with goat lung and liver microsomes were conducted to trap with exogenous glutathione (GSH) the electrophilic intermediate produced via cytochrome P-450-dependent metabolic activation of 3-methylindole (3MI). Microsomal incubation mixtures with [14C]3MI, a NADPH-generating system, and [3H]GSH produced a dual-labeled adduct which was isolated by reverse-phase high-performance liquid chromatography. Reactive 3MI intermediates were also trapped with cysteine. Adduct formation increased in proportion to the concentration of either thiol. Covalent binding of activated 3MI metabolites to microsomal protein was inversely related to adduct production. There were both qualitative and quantitative differences in the formation of GSH adducts by lung and liver microsomes. In the presence of 2 mM GSH, the adduct was produced at a rate of 1.8 nmol/mg protein/min by lung microsomes but only at 0.1 nmol/mg protein/min by hepatic microsomes. The addition of cytosolic fractions containing glutathione S-transferase activity increased GSH adduct formation by approximately 30%. These results support the view that electrophilic 3MI intermediates are trapped by conjugation with GSH, and that organ-selective toxicity is primarily due to much faster rates of cytochrome P-450 oxidation of 3MI in the lung than in the liver.     

The Bushman arrow toxin, diamphidia toxin: Isolation from pupae of Diamphidia nigro-ornata

J. McK. R. Woollard, F. A. Fuhrman and H. S. Mosher. The Bushman arrow toxin, diamphidia toxin: isolation from pupae of Diamphidia nigro-ornata. Toxicon22, 937–946, 1984. — The Bushmen of the Kalahari Desert in Botswana use the pupae of the beetle Diamphidia nigro-ornata Ståhl to poison their arrows. Sequential aqueous extraction, ammonium sulfate precipitation, ultrafiltration and chromatofocusing have given an apparently homogeneous active protein from these pupae with an approximate mol. wt of 54,000, an isolectric point of about 8.0 pH and a lethal potency (minimum lethal dose, mld) between 5 and 20 μg/kg (i.p. mouse). Preliminary pharmacological studies on less purified material show that, after a delay, this diamphidia toxin causes sustained contraction of isolated intestinal smooth muscle. This contraction is not blocked by atropine or mepyramine and, therefore, is not due to release of acetylcholine or histamine. Results on the phrenic nerve - hemidiaphragm preparation demonstrate that in the presence of the toxin, contraction in response to indirect stimulation gradually fails and is accompanied by contracture. Since direct stimulation of the muscle still elicits a contraction, the toxin apparently does not affect the contractile mechanism itself. We conclude that diamphidia pupae contain a protein toxin that is responsible for its lethality. Although this toxin appears to differ in some properties from the toxins reported by Mebs et al., de la Harpe et al. and Kündig, these protein preparations undoubtedly correspond to each other. We did not find any evidence of the low molecular weight toxic component reported by Mebs et al.